Cloning of gene encoding the GP28.5 protein of toxoplasma gondii; peptide fragments of said protein and their applications

ABSTRACT

The invention relates to cloning of the gene for the toxoplasma GP28.5  anen. It also encompasses purified GP28.5 antigen preparations and antigenic polypeptides derived from said antigen, and their applications.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the cloning of the gene encoding a 28.5kDa Toxoplasma excretion-secretion antigen, and to the production ofpeptide fragments representing epitopes thereof, as well as topreparations of said antigen and of its fragments, and to their uses.

2. Description of the Background

Toxoplasmosis is one of the most widespread protozoal infections, bothin man and in animals. It is responsible for about 25% of deaths in AIDSpatients. The congenital infection, the cause of abortions or severeneonatal malformations in man and domestic animals, could be prevented.Indeed, it is known that the primary infection induces a long-lastingimmunity.

In the search for protective antigens permitting the development of avaccine against toxoplasmosis, various antigens have been studied. Theinventors' team was in particular interested in the excretion-secretionantigens (ESA) of the tachyzoites. It has indeed been established,during experiments both in man and in animals, that the ESA antigenswere immunogenic. It was also shown that some ESAs possess epitopes incommon with antigens of bradyzoites. However, bradyzoites are theresistant form of the parasite.

Various approaches, comprising in particular the production ofmonoclonal antibodies, colloidal gold labeling, and molecular biology,led the inventors' team to the characterization of four common antigensCHARIF et al. Exp. Parasitol., 71:117 (1990)!, CESBRON-DELAUW et al.,Proc. Natl. Acad. Sci. USA, 86:7537 (1989)!, DARCY et al., Parasitol.Res. 76:478, (1990)!. The main one, called Gra2 or GP28.5, is aglycoprotein of 28.5 kDa, and it has been shown that it is a constituentof the matrix of the dense granules of tachyzoites, and that it isassociated with the microvilli network of the parasitophorus vacuole ofthe parasite, after invasion of the host.

A 28 kDa antigen (P28), considered as being similar to the GP28.5antigen, has been described by SIBLEY and SHARMA SIBLEY et al. Infect.Immun. 55:2137 (1987)!, and a DNA sequence encoding this antigen hasbeen published by PRINCE et al. Molec. Biochem. Parasitol. 34:3 (1989)!.

SUMMARY OF THE INVENTION

The present invention set itself the aim of producing purifiedpreparations of the GP28.5 antigen, as well as of producing this antigenin recombinant form, and its immunological characterization. Inparticular, the aim of the present invention is the localization and thecharacterization of specific epitopes of the GP28.5 antigen.

The inventors succeeded in obtaining a purified preparation of theGP28.5 antigen, and demonstrated the protective effect of animmunization by this preparation against Toxoplasma gondii infection inmice.

The inventors also cloned the entire gene encoding the GP28.5 antigen,and located the introns and exons, as well as the 5' and 3' noncodingregions.

DETAILED DESCRIPTION OF THE INVENTION

The subject of the present invention is a nucleic acid fragment, whichcomprises a sequence encoding the Toxoplasma GP28.5 antigen. A nucleicacid fragment conforming to the invention is represented in the list ofsequences in the annex under the number SEQ.ID.NO:1. "Sequence encodingthe Toxoplasma GP28.5 antigen" is understood to mean not only the codingsequence identified in the sequence SEQ.ID.NO:1, but also any sequencewhich, taking into account the degeneracy of the genetic code, encodesthe polypeptide represented in the list of sequences in the annex underthe number SEQ.ID.NO:2.

The inventors also showed that the GP28.5 antigen contains several majorepitopes specific for the B cells; one of them, which is recognized by amouse monoclonal antibody called TG17-179 CHARIF et al., Exp.Parasitol., 71:117 (1990)! is located at the C-terminal end of themolecule.

The inventors characterized this epitope and showed that it contained atleast the 5 C-terminal amino acids of GP28.5. In addition, they showedthat this epitope is also a major epitope recognized by human polyclonalantibodies directed against T. gondii.

The invention also encompasses nucleic acid fragments which encodepolypeptides representing epitopes of the GP28.5 antigen.

According to a preferred embodiment of the present invention, saidnucleic acid fragment encodes a polypeptide comprising at least the 5C-terminal amino acids of the sequence SEQ.ID.NO:2.

According to another preferred embodiment of the invention, said nucleicacid fragment encodes a polypeptide comprising fragment 24-129 of thesequence SEQ.ID.NO:2.

According to yet another preferred embodiment of the invention, saidnucleic acid fragment encodes a polypeptide comprising fragment 127-176of the sequence SEQ.ID.NO:2.

The subject of the invention is also recombinant vectors (plasmids,viruses and the like), which comprise at least one nucleic acid fragmentas defined above, encoding the Toxoplasma GP28.5 antigen, or a peptidefragment representing an epitope thereof.

Said vectors are in particular expression vectors, comprising sequencesof the promoter type, terminator type and the like.

The subject of the present invention is also transformed eukaryotic orprokaryotic cells (and in particular microorganisms), which contain atleast one recombinant vector in accordance with the invention.

The subject of the invention is also a polypeptide of 185 amino acidswhose sequence which is represented in the list of sequences in theannex under the number SEQ.ID.NO:2, is that of the Toxoplasma GP28.5antigen.

The invention also encompasses the polypeptides whose sequence differsfrom the abovementioned sequence only by a few amino acids, inparticular polypeptides representing allelic variants or isoforms of theGP28.5 antigen.

The invention also comprises recombinant proteins comprising all or partof the sequence of the SEQ.ID.NO:2 sequence, optionally fused withanother polypeptide sequence; within this framework, the subject of theinvention is in particular:

a recombinant protein of 212 amino acids, comprising the entire sequenceof the GP28.5 antigen;

a recombinant protein comprising amino acids 127-176 of the sequenceSEQ.ID.NO:2;

a recombinant protein comprising amino acids 1-129 of the sequenceSEQ.ID.NO:2;

a recombinant protein comprising amino acids 24-129 of the sequenceSEQ.ID.NO:2.

The invention also relates to a process for producing a recombinantprotein as defined above, which process comprises a step during whichthe transformed cells as defined above, comprising at least one DNAfragment in accordance with the invention, are cultured.

The recombinant polypeptides in accordance with the invention, whenexpressed in E. coli, conserve major epitopes involved in the polyclonalresponse to the GP28.5 antigen, and do so even though the expression inE. coli does not maintain the structural integrity of the GP28.5 proteinwhich is a glycosylated protein.

If it is desired, however, to obtain other epitopes such as epitopes ofcarbohydrate nature, or epitopes corresponding to tertiary andquaternary structures, it will be chosen to produce the recombinantGP28.5 in eukaryotic systems such as for example yeasts orbaculoviruses.

The inventors, in addition, showed that the C-terminal sequence offifteen amino acids of the GP28.5 antigen represents an epitoperecognized by a number of sera from patients suffering from acute orchronic infections, and also located other major B epitopes in thefragment corresponding to amino acids 127-176 of the sequenceSEQ.ID.NO:2. In addition, the inventors located other regions betweenamino acids 24 and 129, and in particular the regions corresponding toamino acids 55-70 and to amino acids 140-160 of the sequenceSEQ.ID.NO:2, which comprise epitopes of the GP28.5 antigen which arerecognized by the human sera.

The subject of the invention is also peptides comprising at least oneepitope of the GP28.5 antigen.

According to a preferred embodiment of the invention, the said peptidecomprises the 5 C-terminal amino acids of the sequence SEQ.ID.NO:2.

According to a preferred arrangement of this embodiment, the saidpeptide comprises between 5 and 15 C-terminal amino acids of thesequence SEQ.ID.NO:2.

According to a preferred feature of this arrangement, said peptidecomprises the 8 C-terminal amino acids of the sequence SEQ.ID.NO:2.

According to another preferred embodiment of the invention, said peptidecomprises amino acids 24-129 of the sequence SEQ.ID.NO:2.

According to another preferred embodiment of the invention, said peptidecomprises amino acids 55 to 70 of the sequence SEQ.ID.NO:2.

According to yet another preferred embodiment of the invention, saidpeptide comprises amino acids 140 to 160 of the sequence SEQ.ID.NO:2.

The sequence of the 5 C-terminal amino acids of the GP28.5 antigen isrecognized in immunotransfer by the mouse monoclonal antibody TG 17-179.A competitive ELISA with longer peptides has shown that theimmunoreactivity was conserved for peptides of 8 residues or more, andlost when the peptide was reduced to the last 6 C-terminal residues orless. Experiments with the octapeptide lacking the C-terminal glutamineresidue showed that it was then 20 times less active. On the other hand,neither the addition of residues to the C-terminal end, nor thesubstitution of the terminal COOH functional group change theimmunoreactivity of the epitope. In addition, competition experimentsbetween the monoclonal antibody TG 17-179 and sera from infectedpatients showed that the epitope defined by this monoclonal antibody isalso a major epitope for the human polyclonal antibodies.

The subject of the invention is also antigenic compositions whichcomprise at least one antigen chosen from the group consisting of:

a purified preparation of GP28.5 antigen;

a polypeptide of sequence SEQ.ID.NO:2;

a fragment of said polypeptide representing an epitope of the GP28.5antigen;

a recombinant protein comprising all or part of the sequenceSEQ.ID.NO:2.

The invention also encompasses a process for preparing polyclonal ormonoclonal anti-GP28.5 antibodies which comprises a step during which ananimal is immunized with an antigenic composition in accordance with theinvention.

The antigenic compositions in accordance with the invention also permitthe preparation of diagnostic reagents or anti-Toxoplasma vaccines. Theinvention also encompasses these reagents and these vaccines.

The present invention will be understood more clearly with the aid ofthe additional description below, which refers to examples relating tothe cloning of the gene encoding the GP28.5 antigen of Toxoplasmagondii, and the identification of the specific epitopes of this antigen.

I) Cloning and Expression of the Gene Encoding GP28.5 of T. gondii

Unless otherwise stated, the techniques for the manipulation of nucleicacids and of molecular cloning which are used in the examples below arethose described by SAMBROOK et al. A Laboratory Manual (second edition),Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)!.

The TG 17-179 antibody was obtained from BALB/C mice immunized with ESAsantigens as described by CHARIF et al. Exp. Parasitol. 71:114 (1990)!.

EXAMPLE 1

Cloning of the Gene Encoding GP28.5 of T. gondii

1) Production of the Toxoplasmas.

Tachyzoites were obtained from the peritoneal fluid of mice infectedthree days earlier with the strain RH of Toxoplasma gondii. Theparasites were harvested in RPMI 1640 medium (GIBCO BRL), filteredthrough polycarbonate membranes (pore diameter 3 microns) (NUCLEPORE)and washed twice in the same medium.

2) Preparation of Toxoplasma gondii nucleic acid. Total RNA was isolatedfrom tachyzoites of Toxoplasma gondii strain RH by extraction withlithium/urea, and the poly(A+) RNA was purified by passing through anoligo(dT)-cellulose column.

3) Construction of the tachyzoites cDNA library.

a) Construction in λgt11

The construction of the Toxoplasma cDNA library in the lambda gt11 phagehas been described by CESBRON-DELAUW et al. CESBRON-DELAUW et al. Proc.Natl. acad. Sci. USA, 86:7537 (1989)!. The library, after amplification,was plated on E. coli Y1090 cells and screened using the monoclonalantibody TG 17-179. The positive clones were detected by incubation withperoxidase-conjugated anti-mouse IgG antibodies, followed by labelingwith 4-chloro-1-naphthol.

Three λgt11 clones were selected: they are clones FM3, FM1 and FM16,containing respectively inserts of 450, 550 and 650 base pairs. EcoRIrestriction fragments of these clones were subcloned into the vectorsM13, mp18 and mp19, and sequenced by the SANGER method FEINBERG et al.Anal. Biochem. 137:266 (1984)!. The sequencing showed that these threeclones all encode the C-terminal end of GP28.5.

b) Construction in λZapII

The tachyzoite cDNA library was constructed in the λZapII vector using"ZAP cDNA SYNTHESIS KIT" (STRATAGENE). The cDNA was synthesized using,as template, 5 μg of polyA RNA from the tachyzoites, with the aid ofreverse transcriptase (MMLVRT) for the synthesis of the first strand,and DNA polymerase I and RNase H for the synthesis of the second strand.After ligation in 3' of an EcoRI adaptor, and after digestion in 5' withXhoI, the excess adaptor was removed by chromatography onacrylamide-agarose (Ac-A 34, IBF). The cDNAs were then ligated into theLacZ gene of 1ZapII ("UNI ZAP XR", STRATAGENE) and encapsulated in vitro("GIGAPAC II GOLD PACKAGING EXTRACT", STRATAGENE). Before amplification,the library comprises 10⁶ recombinant phages.

After amplification, the phages were plated on E. coli XLI-Blue and thelibrary was screened with, on the one hand, the mouse polyclonal serumdirected against the purified GP28.5 antigen (1/100 dilution) and, onthe other hand, the monoclonal antibody TG17-179 (1/500 dilution), bothdiluted in TBS buffer (10 mM TRIS HCl pH 8, 15 mM NaCl). The coinfectionof the bacteria with λZapII and a helper phage (R408, Stratagene) madeit possible to excise the phagemide pBluescript containing the clonedcDNA inserts. The single-stranded DNA obtained from the phagemide in thepresence of the same helper phage was directly used to sequence the cDNAinserts by the dideoxynucleotide method.

The screening of the cDNA library constructed in the λZapII expressionvector allowed the production of longer cDNAs. The longest of them,called "LcDNA" comprises 1100 bp; its sequence in 3' is homologous tothat of the P28 of PRINCE et al., but shorter by at least 121 bp in 5'.

4) Construction of a Toxoplasma gondii genomic library in the phage EMBL3

After partial digestion of the tachyzoite genomic DNA with MboI, thegenomic library was constructed as described by CESBRON-DELAUW et al.Proc. Natl. Acad. Sci., USA, 86:7537 (1989)!.

The library constructed in the phage EMBL3 was plated on E. coli P2392,and screened with the aid of a restriction fragment of the cDNA(EcoRI-SalI fragment of 195 bp), used as probe, after prior labelingwith ³² P. The hybridations were carried out at 65° C., in 5× DENHARDTbuffer 50× DENHARDT buffer contains 1% BSA, 1% PVP, 1% Ficoll (w/v)! and6.6× SSC buffer (10× SSC contains 3M NaCl, 0.3M Na₃ C₆ H₅ O₇.2H₂ O, pH7.2).

A genomic clone called Gra2-EMBL3 was selected; from this clone, a 2040bp fragment, comprising the entire gene as well as 780 bp of 5' flankingsequence and 32 bp of 3' flanking sequence, was subcloned.

FIG. 1 represents the partial restriction map on 5 kb of the genomicclone Gra2-EMBL3, as well as the partial restriction map of a 2040 bpregion thereof, which comprises the entire GP28.5 gene. This figure alsoshows the transcribed region and the region encoding the GP28.5 protein.

Southern blot analysis of tachyzoite genomic DNA hybridized with a 1100bp cDNA probe (clone LcDNA) confirms this restriction map. It alsoindicates that the gene encoding GP28.5 is probably not repeated in thegenome of Toxoplasmas.

The top line is a partial restriction map of the genomic cloneGra2-EMBL3 containing the GP28.5 gene (this gene is indicated by a box).The rest of the figure is a magnification of a 2040 bp region whichrepresents a subclone of the GP28.5 genomic clone, and which containsthe gene encoding GP28.5 as well as the flanking sequences of 780 bp in5' and 32 bp in 3'. The open reading frame of GP28.5 is indicated. TheEcoRI-SalI fragment of 195 base pairs which was used as probe forscreening the libraries is indicated under the restriction maps as wellas the cDNA clones: LcDNA, FM16, FMI, FM3. The sequence AAAA indicatesthe polyA tail of the transcripts. The coding regions are shaded. Therestriction sites are indicated by the following abbreviations: AI=AccI,AII=AvaII; B=Bg1I; E=EcoRV; H=HindIII; N=NaeI; ND=NdeI; PI=PstI;PII=PvuII; S=SalI; SM=SmaI.

The complete sequence of the GP28.5 gene and of the noncoding 5' and 3'regions is shown in the sequence list in the annex under the numberSEQ.ID.NO:1. In order to determine the organization of the GP28.5 gene,the sequence of the genomic library was compared with the sequence ofthe LcDNA clone.

This comparison shows that the GP28.5 gene consists of two exons (5'exon:251 bp; 3' exon:800 bp) separated by a 239 bp intron.

An initiator ATG codon for translation is present in position 886, andthe open reading frame ends at position 1682 with a TAA codon. This openreading frame potentially encodes a protein of 185 amino acids whosetheoretical molecular weight is 19.8 kDa. A potential site of cleavageof a signal sequence is situated between alanines 23 and 24. The openreading frame is bordered in 3' by a noncoding sequence of 32 bp. ANorthern transfer performed on tachyzoite total RNA reveals a singlepopulation of mRNA encoding the GP28.5 protein. The size of themessenger is estimated at approximately 1100 bp, which corresponds tothe size of the LcDNA clone.

In order to define more precisely the site of initiation oftranscription, a primer extension was carried out with tachyzoite totalRNA, using as primer an oligonucleotide (CM10), corresponding topositions 888 to 867 of the sequence. By this method, three differentpotential sites of initiation of transcription were located,respectively at 105, 103 and 102 bp upstream of the signal forinitiation of the translation. The first two may correspond to minorsites of transcription whereas the third is undoubtedly the major siteof initiation of transcription.

The amino acid sequence of GP28.5 is 67 amino acids shorter at itsN-terminal end than the presumed sequence of the P28 antigen previouslypublished by PRINCE et al. In order to verify the fact that the sequenceSEQ.ID.NO:1 is indeed that which encodes the GP28.5 antigen, thecorresponding amino acid sequence was compared to that of five peptidesresulting from the trypsin cleavage of the GP28.5 antigen purified byHPLC as described below. The sequence of these five peptides wascompletely found again in the amino acid sequence deduced from that ofthe open reading frame of the cloned gene.

Example 2

Preparation of Recombinant Proteins Comprising Fragments of the GP28.5Sequence

1) Fusion proteins with Glutathione-S-Transferase

Two cDNA clones representing fragments of the sequence encoding theGP28.5 protein were subcloned into the plasmid pGEX-2T SMITH et al.Gene, 67, 31-40 (1988)). The first clone, FM16, encodes the fifty-nineC-terminal amino acids of GP28.5, and the second clone, L, represents212 amino acids of GP28.5.

5 other fragments (FIG. 3) encoding portions of the GP28.5 protein werealso subcloned into the plasmids pGEX-2T and pGEX-3X (SMITH et al.,cited):

the first clone, ptg.Gra2.1, derived from the LcDNA clone, encodes 212amino acids including the 185 of GP28.5;

the second clone, ptg.Gra2.2, derived from the cDNA clone FM16, encodesthe 59 C-terminal amino acids of the GP28.5 protein;

the third clone, ptg.Gra2.3, encodes 50 amino acids (amino acid 127 toamino acid 176 inclusive, see FIG. 3);

the fourth clone, ptg.Gra2.4, encodes 186 amino acids;

the fifth clone, ptg.Gra2.5, encodes 129 amino acids (N-terminal part ofthe GP28.5 protein, up to amino acid 129 inclusive);

the sixth clone, ptg.Gra2.6, encodes 171 amino acids (from amino acid 15inclusive);

the seventh clone, ptg.Gra2.7, encodes 155 amino acids (from amino acid31 inclusive).

The plasmids pGEX-2T and pGEX-3X containing the DNA inserts were used totransform E. coli JM 109 bacteria. The expression and purification ofthe recombinant proteins as well as of GST alone were carried outaccording to the procedure described by SMITH and JOHNSON (referencepreviously cited). A culture of E. coli JM 109 in the middle of thelogarithmic phase was stimulated with 0.1 mM of IPTG. After growing for1 hour at 37°, the cells were cooled on ice and centrifuged at 4000 rpmfor 15 minutes. The pellet is resuspended in 0.02M PBS, 0.5 mM PMSF, 1mM EDTA, 1% TRITON X-100. The cells are sonicated on ice and centrifugedat 10,000 g for 5 minutes. The recombinant proteins are purified fromthe supernatant, by afffinity chromatography on agarose-glutathione. Theexpression of the recombinant proteins was monitored by immunotransferusing the monoclonal antibody TG17-179 or mouse polyclonal serumdirected against the purified GP28.5 antigen. The recombinant proteinscorresponding to the clones ptg.Gra2.2 (59 amino acids) and ptg.Gra2.3(50 amino acids) were purified with a high yield. On the other hand, therecombinant proteins corresponding to the clones ptg.Gra2.1 (212 aminoacids), ptg.Gra2.4 (186 amino acids) and ptg.Gra2.5 (129 amino acids)were obtained with only low yields which appear to be due to thedegradation of the proteins on the one hand, and on the other, to thepresence of the signal sequence (the first 23 amino acids of the GP28.5protein) in the recombinant proteins.

2) Fusion proteins with β-galactosidase

Recombinant lysogenic phages were produced from the original λgt11 cloneFM16, in E. coli Y1089, according to the process described by HUYNH etal. (Glover, D. M. ed. vol. 1, 49-78 IRL Press, Oxford (1985)!. Crudelysates containing wild-type β-galactosidase or alternatively fusionprotein GP28.5/β-galactosidase! were prepared by inducing cultures atthe logarithmic phase, by heating at 45° C. for 20 minutes and addingisopropyl-β-D-thiogalactopyranoside at a concentration of 10 mM. Afteradditional incubation at 37° C for 1 hour, the culture was concentrated,in a 0.01M Tris buffer, pH8, 0.1M EDTA, 10 mM NaCl, and then lysed bythe addition of 0.25 mg of lysozyme, 0.01M MgCl₂, and 0.2% TRITON X-100,and finally treated with DNAseI at 37° C. for 30 minutes.

The FM1 and FM16 clones react much more strongly with the TG 17-179antibody than the FM3 clone. The restriction map shows that the EcoRIinserts of 450 bp (FM3), 550 bp (FM1) and 650 bp (FM16) containsequences which overlap.

The sequencing of these clones revealed that the reading frames in phasewith that for β-galactosidase were only 17 bp for FM3, and 95 bp and 172bp for FM1 and FM16 respectively, the rest of the inserts correspondingto the nontranslated 3' region plus the polyA tail. It therefore seemsthat the peptide corresponding to the 5 C-terminal amino acids of theGP28.5 antigen encoded by the FM3 clone is sufficient to obtain anantigenic reaction.

II) Purification of the GP28.5 Antigen

Example 3

Purification of the GP28.5 Antigen by HPLC

10¹⁰ tachyzoites of the RH strain were washed twice with 10 mM PBS, pH7.2. After centrifugation at 1000 g for 10 minutes, the pellet wasincubated overnight at 4° C. with gentle stirring, at a concentration of10⁹ parasites/ml in TEN buffer (10 mM TRIS-HCl, pH 7.4; 2 mM EDTA; 150mM NaCl) supplemented with 1% NONIDET P40, 100 U/ml of aprotinin and 40μM PMSF. After centrifugation at 3500 g for 20 minutes, the supernatantwas recovered and filtered on a 0.22 μm MILLIPORE membrane. Thetachyzoite proteins extracted with NP40 were purified by reversed-phaseHPLC chromatography in a C18 VYDAC column (300 Å, particle size: 7 μm,column dimensions: 500×9 mm). 8 ml of crude protein extract were loadedonto the C18 column at 0% of solvent B solvent A consists of 0.5% v/v ofTFA in water; solvent B is a 25/75/0.45 (v/v) mixture ofwater/acetonitrile/TFA!. After 10 minutes of isocratic elution, theproteins are eluted with a 0 to 100% gradient of solvent B, for a periodof 180 minutes, at an elution rate of 2 ml per minute, and detected at215 nanometers. In order to locate the GP28.5 antigen, each HPLCfraction was freeze-dried, and the freeze-dried product was diluted in100 μl of a TEN/PBS mixture 10 mM, pH 8 (v/v)!. 1 μl of each fractionwas tested by dot-blot using the monoclonal antibody TG17-179. 10 μl(which corresponds to about 12 μg of purified protein) of each of thefractions which showed a positive reaction were loaded on an acrylamidegel in the presence of 2-ME. A silver nitrate staining of the gel madeit possible to check their homogeneity and their purity. A similar gelwas prepared and used for the electrophoretic transfer ontonitrocellulose membrane. The antigenicity of the fractions was evaluatedby incubating the membrane with the monoclonal antibody TG17-179. Thedetection was performed with peroxidase-labeled antimouse IgGantibodies.

The major component of the eluate is the GP28.5 protein. The onlycontaminant detected is a 65 kDa protein which is also recognized by theTG17-179 antibody, and which may therefore represent either a dimer ofGP28.5, or a related antigen.

II) Immunological Properties of the GP28.5 Antigen and its Fragments

Example 4

Immunization of Mice and Protection Trials

8 to 10-week old female OF1 mice were immunized in the following manner:forty-two, thirty-five, twenty-seven and seven days before theinfection, the following antigens:

ESA corresponding to the secretion of 5×10⁷ tachyzoites; oralternatively,

15 μg of GP28.5 purified by HPLC as described in Example 3 weresuspended in 100 μl of PBS, emulsified with 100 μl of incompleteFREUND's adjuvant and administered by subcutaneous injection.

Control mice were treated according to the same procedure with PBSsupplemented with 100 μl of incomplete FREUND's adjuvant.

On the day of the infection, the sera were collected, then a suspensionof 1200 cysts in 0.3 ml of PBS, pH 7.2, was administered orally.

The sera from mice immunized with ESA or GP28.5 purified by HPLC weretested by immunoelectrophoretic transfer, against tachyzoite ESAantigens extracted with NP40, in order to determine if they recognizethe GP28.5 antigen.

It appears that the immunoreactivity profile is essentially the samewhether the visualization is made with mouse immunoserum or with themonoclonal antibody TG 17-179: in both cases, three bands of about 100,65 and 28 kDa are visualized. The two bands with the highest molecularweight which are recognized by the TG 17-179 antibody probably representGP28.5 polymers.

FIG. 2 represents the percentage of mice surviving after the infection.This figure shows that, whereas only 25% of the control mice immunizedsolely with incomplete FREUND's adjuvant survive 15 days after theinfection, 75% of the mice immunized with the GP28.5 antigen purified byHPLC are still alive 50 days after the infection. When the mice areimmunized with the ESA antigens, 45% survive 50 days after theinfection. These results show that the immunization with theHPLC-purified GP28.5 antigen leads to a significant protection.

Example 5

Reactivity of Human Sera with the Recombinant Proteins and Recognitionof the C-Terminal Epitope

The human sera were obtained from patients carrying a chronic Toxoplasmagondii infection (TG positive). Sera from healthy patients were used ascontrol (TG negative). The test was carried out by ELISA, and thevisualization made using biotinylated anti-human IgG antibodies; thebinding of these antibodies was detected using peroxidase-labeledstreptavidin-biotin complexes. The peroxidase substrate used is OPD andthe optical density was measured at 492 nm and expressed with respect toa control done using as antigen GST, according to the following formula:

OD=OD of the serum reacting with the recombinant protein of 59 aminoacids--OD of the serum reacting with GST. Under these conditions, themean optical density of the TG-negative sera was 0.23. 74% of thefifty-nine TG-positive sera were above this optical density. Thecorrelation with a conventional test of the anti-TG antibodies is:R=0.74 P.

In spite of this correlation, patients having 50 to 100 IU/ml of anti-TGIgG show very dispersed values for levels of antibodies against the FM16recombinant protein (the optical densities range between 0.35 and 1.5).It therefore seems that a homogeneous level of anti-TG antibodies can infact reflect varying responses to the GP28.5 antigen.

Ten sera which are negative by ELISA against the protein containing thefifty-nine amino acids were tested by immunotransfer against the fusionprotein containing the 212 amino acids (LcDNA clone). Eight of thesesera react with the protein comprising the 212 amino acids, whereas 7 ofthe control sera react negatively. It is probable that these 8 serarecognize epitopes present on the protein of 212 amino acids, and nearerthe N-terminal end than those carried by the protein comprising thefifty-nine amino acids.

Th binding of TG 17-179 to the recombinant antigen FM16 was tested bycompetitive ELISA in the presence of sera from infected patients; theserum was added at a dilution of 1/100 to the solution of monoclonalantibodies.

These competition trials between TG 17-179 and sera from infectedpatients show that the human polyclonal antibodies react with theC-terminal epitope of GP28.5: among the 12 human sera tested, 10 inhibitat varying degrees the binding of TG 17-179 to the FM16 fusion protein.

The reactivity of the fusion proteins produced by the FM1 and FM3 cloneswith the TG 17-179 antibody was also tested; FM1 and FM16 react muchmore strongly with the TG 17-179 antibody than the FM3 clone.

Now, the sequencing of these clones has revealed that the reading framein phase with that for β-galactosidase, is only 17 bp for FM3, and 95 bpand 172 bp for FM1 and FM16 respectively. It therefore appears that thepeptide encoded by the FM3 clone, and corresponding to the 5 C-terminalamino acids of the GP28.5 antigen is sufficient in order to obtain anantigenic reaction.

However, since it also appeared that the binding of the monoclonalantibody TG 17-179 to this FM3 peptide is weaker than the binding ofsaid antibody to the peptides encoded by the longer clones FM1 and FM16,the inventors undertook to determine the optimal length of the epitope.

Example 6

Immunoreactivity of Synthetic Peptides Corresponding to the C-TerminalEnd of GP28.5

Peptides overlapping and covering the carboxy-terminal sequence ofGP28.5 (1 to 15 residues) were synthesized, using the MERRIFIELD method.

They were tested by competitive ELISA with the FM16 fusion protein.

The fifteen terminal amino acids of GP28.5 contain the epitope reactingwith the monoclonal antibody TG 17-179. The capacity of this peptide toinhibit the reactivity in ELISA against the recombinant proteincontaining the fifty-nine amino acids, of sera obtained from patientssuffering from acute or chronic infections was tested. Both types ofsera (acute and chronic) are inhibited, to a varying degree, by theC-terminal peptide of fifteen amino acids. For the chronic sera, thepercentage of inhibition varies from 8 to 100% and no correlation isobserved with the starting optical density. The low level of inhibitionobserved for some sera may be due to the presence of antibodies with lowaffinity for this peptide. However, even on multiplying by ten theconcentration of the peptide, the percentage inhibition is notincreased. The specificity of the inhibition was demonstrated using ascontrol a peptide with a different sequence and similar length. Thepercentage inhibition of the acute sera varies from 15 to 90% and isalso independent of the starting optical density.

The inhibition studies carried out therefore show that the peptide of 15amino acids comprises a major epitope of the C-terminal region offifty-nine amino acids, for four out of twelve of the sera obtained fromchronic patients, and for three out of twelve of the sera obtained frompatients suffering from acute infections. In addition, three out oftwelve of the chronic sera, and four out of twelve of the acute sera,show a partial inhibition (35-80% inhibition).

It therefore appears that the polyclonal response to the GP28.5 antigeninvolves a reactivity with the fifteen C-terminal amino acids, areactivity of which the degree varies according to the individual. Onthe other hand, the degree of this response apparently does not varybetween the acute infection and the chronic infection, no difference inthe average percentage inhibition for each of the two groups having beenobserved.

However, five out of twelve chronic sera and five out of twelve acutesera which react with the C-terminal region of fifty-nine amino acids ofGP28.5 do not react with the peptide of fifteen C-terminal amino acids,which shows that other major B epitopes exist in this region offifty-nine amino acids and especially in the fragment corresponding toamino acids 127-176 of the sequence of the GP28.5 antigen.

In the case of C-terminal peptides having less than 15 amino acids,results are the following:

The strongest inhibition of the binding of the monoclonal antibody TG17-179 to the fusion protein is obtained with peptides comprisingbetween 11 and 15 residues.

The inhibition is however obtained with the octapeptide comprising the 8C-terminal residues.

The binding of TG 17-179 to the C-terminal heptapeptide is 8 timesweaker than its binding to the octapeptide. Finally, theimmunoreactivity of the synthetic peptide is lost when it comprises 6C-terminal amino acids or less.

The octapeptide lacking its COOH functional group and the octapeptidecomprising two additional C-terminal residues (alanine) were alsotested. These two peptides have practically the same activity as theC-terminal octapeptide: this shows that the carboxyl functional group isnot necessary for the immunoreactivity of the epitope recognized by TG17-179.

The same peptides were tested by direct ELISA. Under these conditions,the immunoreactivity as a function of the length of the peptidedecreases more rapidly: it is indeed lost for peptides of less than 10residues in length. Consequently, it appears that by direct ELISA, themonoclonal antibody requires a longer peptide than in the inhibitiontest. In this particular case, it is unlikely that this is due to a poorabsorption of the shortest peptides onto the microtiter plate, given thehydrophobic nature of the amino acids of said peptides.

Peptides lacking C-terminal residues were also synthesized: whereas theincubation of TG 17-179 with the synthetic octapeptide covering theentire C-terminal residues inhibits the binding of the recombinantfusion protein, the corresponding heptapeptide lacking thecarboxy-terminal glutamine residue is 64 times less active ascompetitor, and the hexapeptide lacking the two carboxy-terminalresidues is 104 times less active.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2152 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: join(886..1035, 1275..1682)                                     (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 886..1035                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 1275..1682                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: intron                                                          (B) LOCATION: 1036..1274                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CTGCAGTCTTAATCATTTCCACATAGTTTTTGTTCCCCCAGACAATCAATCCTGGCTGAG60                CCCCCCATGTACGCGTTACCACCTGCCAGTGCATATGGGTTTGCATATTTTTGCTGAAGT120               CCGAAAGAGGGGCCACGCAAAACGTTACCGGTTTGCTGCAGGCAGCCAGGTAGGTGGAAC180               AGATCTCCAGCGAGTACGACCACTGTGCGTGTACTTACGCCAAAAGGAAAATACACCTGC240               ACCGCTATTAAGCGGCAGTCGTCTACCTGAATCGTCTCCCCGGCCTCATCATTTTGTTCG300               ACACAAGTTTCCATTAGGACTTTGTGACAGTCGTCTGCTCTCGACCTTCCAACCGTCTCG360               TGGACGAAAAATCCTCGGGTGACTTGCCGTGGACGAACGCCTCCCGTTTGCTTCTACAAG420               TGACTATGCGATAGGTTCCGCAGTGCAGCCAGGCTTGCGAAAAACAAGTTCGTCGCAAAA480               GGTTAATTACCTACGCACCACGAAGGAAAACGCGTATCACGTCAGTCCTTACGGTCAATA540               TACAAATACTTGGCCGTCCAGTGGAGGCAACGTGCCCGTCGCACGGTGATACTGACTGGT600               GACTTGCACGTACGTCTCTCGCCGGCGTCCAAACCAAATTGACCCGGGGCAGCCTACTCC660               CTGTCGTCCCTTAGGCTAAGTGCGAGCAACATCTCTACACAGAGACGACGCCAGAGACGC720               AAAATGAACAGCGGAACCTGCGTCGCTGTCTGTCCTGCGAACTGATGACAGAAAGGGTCA780               TTAAACGATTTCTTTTGCAATTCGCGTCGTTATCGCACGTTGTTTCTCTTCCCACGAATA840               GTTGTTTTGATTAGATATTGCTTCTTCTCCACATATCGCCTCACAATGTTCGCC894                     MetPheAla                                                                     GTAAAACATTGTTTGCTGGTTGTTGCCGTTGGCGCCCTGGTCAACGTC942                           ValLysHisCysLeuLeuValValAlaValGlyAlaLeuValAsnVal                              51015                                                                         TCGGTGAGGGCTGCCGAGTTTTCCGGAGTTGTTAACCAGGGACCAGTC990                           SerValArgAlaAlaGluPheSerGlyValValAsnGlnGlyProVal                              20253035                                                                      GACGTGCCTTTCAGCGGTAAACCTCTTGATGAGAGAGCAGTTGGG1035                             AspValProPheSerGlyLysProLeuAspGluArgAlaValGly                                 404550                                                                        TAAGTTGGCAAAAGTAATGATAGAGGCAGGGGTTGAACGATAGGCGGCTGCAGATTTGTA1095              TAACACAACATGATGTAGCTGCCACGGTTTTTTTTCGGAGAGTGATGCCGTCTGACTGTC1155              ATCGCACCCATGGGAGCTAGGGAGGTGCGCTTCTGTGTGATATGTATTGTCCTAGTCCAA1215              TTTCCCACGCACTGTAGTGTCTTGAGACTCGGTGCCATGTAGAATTTTGTGTCTGCAGA1274               GGAAAAGGTGAACATACACCACCACTCCCAGACGAGAGGCAACAAGAG1322                          GlyLysGlyGluHisThrProProLeuProAspGluArgGlnGlnGlu                              556065                                                                        CCAGAAGAACCGGTTTCCCAACGTGCATCCAGAGTGGCAGAACAACTG1370                          ProGluGluProValSerGlnArgAlaSerArgValAlaGluGlnLeu                              707580                                                                        TTTCGCAAGTTCTTGAAGTTCGCTGAAAACGTCGGACATCACAGTGAG1418                          PheArgLysPheLeuLysPheAlaGluAsnValGlyHisHisSerGlu                              859095                                                                        AAGGCCTTCAAAAAAGCAAAGGTGGTGGCAGAAAAAGGCTTCACCGCG1466                          LysAlaPheLysLysAlaLysValValAlaGluLysGlyPheThrAla                              100105110                                                                     GCAAAAACGCACACGGTTAGGGGTTTCAAGGTGGCCAAAGAAGCAGCT1514                          AlaLysThrHisThrValArgGlyPheLysValAlaLysGluAlaAla                              115120125130                                                                  GGAAGGGGCATGGTGACCGTTGGCAAGAAACTCGCGAATGTGGAGAGT1562                          GlyArgGlyMetValThrValGlyLysLysLeuAlaAsnValGluSer                              135140145                                                                     GACAGAAGCACTACGACAACGCAGGCCCCCGACAGCCCTAATGGCCTG1610                          AspArgSerThrThrThrThrGlnAlaProAspSerProAsnGlyLeu                              150155160                                                                     GCAGAAACCGAGGTTCCAGTGGAGCCCCAACAGCGGGCCGCACACGTG1658                          AlaGluThrGluValProValGluProGlnGlnArgAlaAlaHisVal                              165170175                                                                     CCCGTCCCAGACTTTTCGCAGTAATGTTGACTACGACGAAAGTGATGCGCAGGC1712                    ProValProAspPheSerGln*                                                        180185                                                                        TGGAAAGCCGCTGAAGGGAGAAGTCTACAAAGCCGATCAGTGAAAAATGTGTGGGGAGGT1772              GGTCTTGTTGCAGGAATGCAATGTGTTAAGCATCGTGTTCGAATGCAGTGCGTGTATCAG1832              TTGTGCGCGGAAGGACACTGCTTCAATGTTAAGAACCTGTTTTCTCCGTAGAGAGGACCA1892              AAAGACGATTGCAAAACTGGTATGTACGCAATAGCCCAATGCCGGACGTCAGTTGGTTGT1952              ATGTGACGCTCCCAGATGTCATATGCCTTGTGAGTGTGTCTGGGATGCAAGTTTTTGGTG2012              TGCGTTGATTTCGCCAGCTTATGACAGTGGCAGACGAATTATTGACATGATACAAGGACG2072              CAGAAAGGAACAAACACCGTAGTTCCAGTCGACACAGAAAGGGAGGGTAAAGAAAGTAAT2132              TGAAAGGTGATTTTAGATAA2152                                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 185 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetPheAlaValLysHisCysLeuLeuValValAlaValGlyAlaLeu                              151015                                                                        ValAsnValSerValArgAlaAlaGluPheSerGlyValValAsnGln                              202530                                                                        GlyProValAspValProPheSerGlyLysProLeuAspGluArgAla                              354045                                                                        ValGlyGlyLysGlyGluHisThrProProLeuProAspGluArgGln                              505560                                                                        GlnGluProGluGluProValSerGlnArgAlaSerArgValAlaGlu                              65707580                                                                      GlnLeuPheArgLysPheLeuLysPheAlaGluAsnValGlyHisHis                              859095                                                                        SerGluLysAlaPheLysLysAlaLysValValAlaGluLysGlyPhe                              100105110                                                                     ThrAlaAlaLysThrHisThrValArgGlyPheLysValAlaLysGlu                              115120125                                                                     AlaAlaGlyArgGlyMetValThrValGlyLysLysLeuAlaAsnVal                              130135140                                                                     GluSerAspArgSerThrThrThrThrGlnAlaProAspSerProAsn                              145150155160                                                                  GlyLeuAlaGluThrGluValProValGluProGlnGlnArgAlaAla                              165170175                                                                     HisValProValProAspPheSerGln                                                   180185                                                                        __________________________________________________________________________

We claim:
 1. An isolated nucleic acid fragment encoding a polypeptidecomprising at least one epitope of Toxoplasma GP28.5 antigen, whereinthe fragment is selected from the group consisting of(b) a nucleic acidfragment encoding a polypeptide consisting of amino acids 1 to 129 ofSEQ ID NO: 2, (c) a nucleic acid fragment encoding a polypeptideconsisting of amino acids 24 to 129 of SEQ ID NO: 2, (d) a nucleic acidfragment encoding a polypeptide consisting of amino acids 55 to 70 ofSEQ ID NO: 2, and (e) a nucleic acid fragment encoding a polypeptideconsisting of amino acids 140 to 160 of SEQ ID NO:
 2. 2. A recombinantvector comprising at least one nucleic acid fragment as claimed inclaim
 1. 3. A eukaryrotic or prokaryotic host cell transformed with atleast one recombinant vector as claimed in claim
 2. 4. A method ofproducing a polypeptide comprising at least one epitope of theToxoplasma GP28.5 antigen, comprising culturing the transformed cellaccording to claim 3 to produce the polypeptide in the cell culture,followed by isolating the polypeptide from the cell culture.
 5. Anisolated nucleic acid encoding a fusion polypeptide comprising afragment of Toxoplasma GP28.5 antigen comprising at least one epitope ofthe Toxoplasma GP28.5 antigen fused to a heterologous polypeptidesequence, wherein the fragment of the Toxoplasma GP28.5 antigen isselected from the group consisting of(b) a fragment consisting of aminoacids 1 to 129 of SEQ ID NO: 2, (c) a fragment consisting of amino acids24 to 129 of SEQ ID NO: 2, (d) a fragment consisting of amino acids 55to 70 of SEQ ID NO: 2, and (e) a fragment consisting of amino acids 140to 160 of SEQ ID NO:
 2. 6. A recombinant vector comprising at least onenucleic acid fragment as claimed in claim
 5. 7. A eukaryotic orprokaryotic host cell transformed with at least one recombinant vectoras claimed in claim
 6. 8. A method of producing a fusion polypeptidecomprising at least one epitope of the Toxoplasma GP28.5 antigen,comprising culturing the transformed cell according to claim 7 toproduce the fusion polypeptide in the cell culture, followed byisolating the fusion polypeptide from the cell culture.